Some environmental toxicants are alkylating agents that can damage DNA. If left un-repaired, DNA alkylation damage can lead to mutation, cell death, and can promote carcinogenesis. Fortunately, most cells can prevent the detrimental effects of alkylating agents by initiating cell-signaling programs that promote the repair of damaged DNA. Our long-term research goals are to identify and characterize signaling pathways used to respond to DNA alkylation damage, and to understand the role of these damage-signaling pathways in exposure-related diseases. We have computationally identified a novel environmental stress code (ESC) that (1) is a gene-specific codon usage pattern and (2) is predicted to help regulate cellular responses to DNA alkylation damage. We hypothesize that the identified ESC works in conjunction with tRNA modifying enzymes to promote the translation of DNA repair, signaling, and stress tolerance proteins after damage. Further, we hypothesize that one of the enzymes controlling this program of enhanced translation is tRNA methyltransferase 9 (Trm9). In this study, we will test the novel hypothesis that in response to alkylation damage, Trm9 catalyzes the methylation of select tRNA molecules to enhance the translation of a network of damage-response proteins. We will exploit three powerful model systems (based in Saccharomyces cerevisiae, humans, and mice) to test our hypothesis, to increase our understanding of translational regulation after DNA damage, and to correlate our results to humans. Specifically, we will determine the damage-induced substrate specificity of S. cerevisiae Trm9 (Specific Aim 1) in order to define the spectrum of tRNA molecules enzymatically methylated in response to alkylation damage. In addition, we will use codon-specific reporter constructs and a TAP-tagged protein library to determine how the ESC and Trm9 influence the translation of a damage response network. Experiments proposed in Specific Aim 1 exploit the power of systems-based studies in S. cerevisiae to characterize global protein networks. We will use HEK293 cell models to characterize the damage-induced substrate specificity of human Trm9 (Specific Aim 2) and to analyze the role of Trm9 in the translation of codon-specific reporter constructs. Excitingly, we have linked human Trm9 activity to the regulation of specific DNA repair proteins (Rad54, Brca2, Bccip, Ercc8, Tdg, Msh4, Rad51, and DNA Ligase IV), and we will study how Trm9 affects their damage-induced translation. Importantly, Specific Aim 2 will allow us to use a human system to model the activity of a novel damage-response protein. We will use mouse cell systems (Specific Aim 3) to characterize the substrate specificity of mouse Trm9. In addition, we will study the regulation of Trm9 in developing mouse embryos and adult organs, and use organ extracts to determine tRNA methylation patterns in vivo. Finally, we will use available Gene Trap cell lines to generate a knockout model of Trm9, and we will determine the viability of resulting mice. We will use Specific Aim 3 experiments to build an animal model for future studies. Ultimately, we will study the relationship between DNA alkylation damage and protein translation, and determine how RNA based signals coordinate DNA damage response pathways. In the future, we can use RNA modifications as biomarkers for DNA alkylation damage and environmental exposures. Additionally, damage-signaling mechanisms are potential targets for therapies related to cancer treatment.